Download Ligand Exchange Chromatography

III / CHIRAL SEPARATIONS / Ligand Exchange Chromatography
Karlsson A and Karlsson O (1997) Enantiomeric separation
of amino alcohols using Z-L-Glu-L-Pro or Z-L-Glu-D-Pro
as chiral counter ions and Hypercarb as the solid phase.
Chirality 9: 650}655.
Knox JH and Jurand J (1982) Separation of optical isomers
by zwitterion pair chromatography. Journal of Chromatography 234: 222}224.
Knox JH and Ross P (1997) Carbon-based packing materials for liquid chromatography. Advances in Chromatography, 74}162.
2369
Petterson C and Heldin E (1994) A practical approach
to chiral separations by liquid chromatography. In:
Subramanian G (ed.) Ion-Pair Chromatography in
Enantiomer Separations, pp. 279}310. Weinheim:
VCH.
Schill G, Wainer IW and Barkan SA (1986) Chiral separation of cationic drugs on an 1-acid glycoprotein
bonded stationary phase (Enantiopac威). Journal of
Chromatography 365: 73}78.
Ligand Exchange Chromatography
V. A. Davankov, Nesmeyanov-Institute of OrganoElement Compounds, Russian Academy of Sciences,
Moscow, Russia
Copyright ^ 2000 Academic Press
Introduction
Ligand exchange chromatography (LEC) was Rrst
introduced by Helfferich in 1961 as a general chromatographic technique to separate compounds which
are able to form labile complexes with transition
metal cations. The basic idea was to immobilize such
ions as Cu(II) or Ni(II) on a stationary phase, in
particular, a cation exchanger with sulfonic or
carboxylic functional groups, and then let the ions
form labile coordination compounds with analytes
which possess electron-donating functional groups
and can enter the coordination sphere of the metal
ion, thus acting as ligands. Those analytes that form
stronger complexes with the central ion, are retained
longer on such a metal ion-incorporating stationary
phase.
Starting with the idea that, in the densely packed
coordination sphere, ligands enter multipoint interactions with each other and should therefore mutually recognize the spatial conRguration of neighbours, Davankov and Rogozhin (1968}71) synthesized chiral complex-forming resins and further
developed LEC into a powerful chiral chromatographic method. This technique, for the Rrst time in
liquid chromatography, resulted in a complete and
reliable resolution of a racemate into constituent enantiomers. Typical analytes for enantioseletive LEC
are members of such important classes of chiral compounds as amino acids, hydroxy acids and amino
alcohols. Having soon become one of the most extensively investigated methods for the direct resolution
of enantiomers, LEC maintained for a long period of
time its leading positions in developing novel chiral
chromatographic systems and evaluating mechanisms
of chiral recognition and discrimination. This technique contributed much to the successful development of chiral silica-bonded stationary phases, both
monomeric and polymeric, chiral coatings on column-packing materials and chiral mobile phase additives. In this last technique, it transpired that
the central metal ion does not need to reside in the
stationary phase, but, instead, can be a part of a
chiral complex that is doped into the mobile phase to
interact there with the analytes. This widens signiRcantly the deRnition of LEC to a chromatographic
process in which the formation and breaking of labile
coordinate bonds to a central metal cation are responsible for the separation of complex forming
analytes.
Ligand exchange has been realized in all known
modes of enantioselective chromatography, including
liquid chromatography, thin layer chromatography,
gas chromatography, capillary electrophoresis and
countercurrent liquid chromatography. In gas
chromatography the principle of ligand exchange is
more commonly known under the name ‘complexation chromatography’.
Theoretical Aspects of Chiral
Discrimination in LEC
Being built of protons, neutrons and electrons, which
basically are chiral elementary species, all atoms
possess inherent chirality. Therefore, two enantiomeric molecules resulting from a mirror-symmetric
arrangement of a certain ensemble of atoms in
space, can differ in their thermodynamic stability.
However, this difference may only amount to about
10\14 J mol\1, which practically makes the enantiomers energetically identical and indistinguishable in
any achiral environment. Using any chromatographic
technique, two enantiomers can be recognized and
2370
III / CHIRAL SEPARATIONS / Ligand Exchange Chromatography
discriminated by the action of a chiral selector only.
The latter has to be involved in interaction with the
enantiomers, resulting in the formation of adducts
which, being diastereomeric species, may differ in
their stability. These adducts in LEC are ternary complexes composed of the molecule of the chiral selector, the complexing metal ion, and the enantiomer to
be recognized. Thermodynamic enantioselectivity of
the system, i.e. the difference in stability of the above
two diastereomeric ternary complexes which incorporate (R) or (S) enantiomers of the analyte, represents the sole source of chiral discrimination of the
enantiomers in LEC. For an analytical scale resolution of enantiomers, the enantioselectivity does not
need to be high, since, in principle, enantioselectivity
of G0"25 J mol\1 would correspond to a separation factor "1.01, which is fully sufRcient for two
peaks to be resolved in a gas chromatographic capillary column generating about 105 theoretical plates.
For a preparative scale liquid chromatography, one
would prefer to deal with a separation factor of at
least "1.5, which would correspond to enantioselectivity of about 1 kJ mol\1. Ligand exchange
often generates a discrimination effect of this order of
magnitude. With the G0 increasing further, the
column selectivity, , rises exponentially, according
to the general relation G0"RT ln . In LEC systems with polydentate ligands, separation selectivities
of up to "10 are known.
As a matter of principle, a chiral selector has to
enter into at least a three-point interaction with the
enantiomers, in order to be able to recognize the
difference in the spatial structure of the latter. This
requirement is easily met with tridentate ligands
forming ternary complexes with metal ions which
have a coordination number of six. Among practically important classes of organic compounds, however, are molecules with two electron-donating
heteroatoms, N and/or O, situated in a chain of
carbon atoms in positions 1,2 or 1,3, e.g. in 1,2- and
1,3-diamines, 1,2- and 1,3-amino alcohols, - and amino acids and - and -hydroxy acids. When forming complexes with metal ions, these compounds
usually function as bidentate ligands with both heteroatoms coordinated to the same metal ion in the
form of a Rve- or six-membered chelate ring.
It is very important that formation and dissociation
of the bonds in the coordination sphere of the metal
ion be fast. Otherwise, a slow rate of ligand exchange
would degrade the efRciency of the chromatographic
system. Therefore, only metals forming kinetically
labile complexes can be employed in LEC, preferably
Cu(II), Ni(II) and Zn(II). Even with the above metal
ions, it has been repeatedly reported that the efRciency of ligand exchanging systems can be enhanced by
raising the column temperature to about 503C, which
speeds up the ligand exchange. Ions of Cr(III),
Co(III), Pd(II) and the like, form kinetically inert,
stable complexes unsuitable for LEC. However, the
second coordination sphere of these complexes,
which actually is their solvation shell, is well organized, too, and it does exchange its ligands readily, so
that an enantioselective chromatography process can
also be based on outer coordination sphere ligand
exchange.
Zn(II) would usually coordinate four heteroatoms
with lone electron pairs in the form of a tetrahedron.
The coordination number of the Ni(II) ion is six and
its ligands are usually arranged in an octahedron.
Copper binds four ligands in its main coordination
square, but offers two additional, more remote
axial positions, so that a complex with the coordination number six adopts a distorted octahedral
structure.
It is worth noting, that the optical purity of a complex-forming chiral selector employed in an LEC system does not need to be necessarily 100%. It is
a distinguishing feature of all direct chromatographic
separations of enantiomers that even a selector contaminated by its enantiomeric form can provide
a complete resolution of racemic analytes. Enantiomeric impurities in the chiral selector do not diminish column efRciency but merely result in the two
sharp enantiomeric peaks of the analyte approaching
each other and completely coalescing into one sharp
peak when the enantiomeric excess (e.e.) of the selector falls to zero. This phenomenon (which is not an
issue, at all, with proteins or natural carbohydrates
as the chiral selectors, since these selectors are available in only one enantiomeric form) has been repeatedly proven experimentally in chiral exchanging systems involving amino acids of known e.e. as the
selector.
Chiral-Bonded Ligand Exchanging
Stationary Phases
The Rrst chiral ligand exchanging polystyrene-type
stationary phases were synthesized and tested by
Rogozhin and Davankov as early as 1968, while
analogous silica-bonded phases emerged a decade
later. Even nowadays, cross-linked polystyrene resins
remain popular in preparative chromatography,
mainly due to their excellent chemical durability and
high loading capacity. To prepare such chiral packings, spherical particles of styrene-divinylbenzene
copolymers are usually subjected to chloromethylation and the active chlorine atoms are then replaced
by amino groups of chiral natural -L-amino acids,
in particular (S)-proline and (S)-hydroxyproline.
III / CHIRAL SEPARATIONS / Ligand Exchange Chromatography
2371
(According to modern stereochemical nomenclature,
L- and D--amino acids belong to R and S conRguration series, respectively, with a few exceptions, however.) The structure of a chiral selector immobilized
in this way as well as its complex with a Cu(II) ion
and an amino acid analyte, both of which bind to the
selector during the process of LEC, can be represented by the following scheme:
tate amino acid-type ligands, like histidine or allohydroxyproline, the opposite elution order of the
enantiomers is observed, thus giving values smaller
than 1. Many other types of chiral selectors have been
similarly immobilized on chloromethylated crosslinked polystyrene beads, with the result that cyclic
amino acids as well as benzyl-substituted chiral propan-1,2,-diamine display the highest selectivity and
largest application range. Typical examples for resolving racemates of common -amino acids on these
phases are presented in Table 1.
Two additional polymeric matrixes, cross linked
polyacrylamide and poly(glycidylmethacrylate), have
also been shown to be useful for immobilizing chiral
amino acid selectors. The mode of functionalizing of
these resins, when they incorporate (S)-phenylalanine
and (S)-proline as typical chiral selectors, can be represented by the following structures:
Here, the most important features of the ternary mixed-ligand bis(amino acidato)copper complex are:
The Rrst resin demonstrates higher afRnity to (R)isomers of all amino acids, without any exception,
resolving all racemates with a selectivity of at least
"1.3.
All the above polymeric ligand exchangers have
been used successfully for both analytical and preparative resolution, e.g. for obtaining highly radioactive tritium-labelled optically active amino acids. In
order to remove trace copper ions from enantiomers
resolved in preparative experiments, the fractions of
interest can be gravity Rltered through a small additional column packed with silica, any chelating resin
or even the same chiral resin, with a blue band of
Cu(II) gradually forming at the top of the subsidiary
column.
Silica-bonded chiral ligand exchangers were expected to demonstrate in analytical-scale separations
still better column efRciency than polymer gels. Three
types of bonded phases were suggested independently
and simultaneously in 1979 by the groups of
Foucault, Davankov and Guebitz, based on three
different ways of activating the initial macroporous
E the formation of two Rve-membered chelate rings;
E the occupation of the four positions of the Cu(II)
ion main coordination square by four electrondonating atoms of the ligands;
E the trans-arrangement of the two negatively
charged carboxyl groups and the bi-substituted nitrogen atoms, respectively, which minimizes both
the electrostatic and sterical repulsion of the
ligands in the complex.
The resins incorporating (S)-proline and (S)-hydroxyproline, when loaded with Cu(II) ions and
eluted with ammonia solution, are usually found to
retain -amino acids of the opposite, (R)-conRguration more effectively. In this case, the enantioselectivity of the system, when expressed as the ratio of
retention factors of the (R)- and (S)-enantiomers of
the analyte, "kR/kS, is larger than 1. With triden-
2372
III / CHIRAL SEPARATIONS / Ligand Exchange Chromatography
Table 1 Enantioselectivity, "kD/kL, of resolution of racemic amino acids on the Cu(II) forms of polystyrene resins which incorporate
residues of L-proline, L-hydroxyproline, L-allo-hydroxyproline, L-azetidine carboxylic acid and N1-benzyl-(R)-propane-1,2-diamine.
Mobile phases: aqueous ammonia solutions. Ambient temperature
Racemic amino acid
RPro
RHyp
RaHyp
RAzCA
RBzpn
Alanine
Aminobutyric acid
Norvaline
Norleucine
Valine
Isovaline
Leucine
Isoleucine
Serine
Threonine
Allothreonine
Homoserine
Methionine
Asparagine
Glutamine
Phenylglycine
Phenylalanine
-Phenyl -alanine
Tyrosine
Phenylserine
Proline
Hydroxyproline
Allo-hydroxyproline
Azetidine carboxylic acid
Ornithine
Lysine
Histidine
Tryptophan
Aspartic acid
Glutamic acid
1.08
1.17
1.34
1.54
1.29
}
1.27
1.50
1.09
1.38
1.55
}
1.04
1.18
1.20
1.67
1.61
}
2.46
}
4.05
3.85
0.43
}
1.0
1.10
0.37
1.40
0.91
0.62
1.04
1.22
1.65
2.20
1.61
1.25
1.70
1.89
1.29
1.52
1.45
1.25
1.22
1.17
1.50
2.22
2.89
1.07
2.23
1.82
3.95
3.17
0.61
2.25
1.0
1.22
0.36
1.77
1.0
0.82
1.04
1.18
1.42
1.46
1.58
}
1.54
1.74
1.24
1.48
}
}
1.52
1.20
1.40
1.78
3.10
}
2.36
}
1.84
1.63
1.48
}
1.20
1.33
1.32
1.10
0.81
0.69
1.06
1.29
1.24
1.40
1.76
}
1.24
1.68
2.15
0.78
}
}
1.29
1.44
1.25
1.38
1.86
}
1.78
}
2.48
2.25
1.46
}
1.0
1.06
0.56
1.13
0.88
0.77
0.70
0.49
0.48
0.49
0.31
}
0.49
0.62
0.62
0.44
}
}
0.60
0.70
}
0.49
0.52
0.38
}
}
0.47
}
}
}
1.0
1.0
0.85
}
1.0
}
microparticulate silica gel, namely, by Rrst grafting
silica with aminopropyl, chloroalkyl, and 3glycidoxypropyl groups, respectively:
Whereas the aminopropyl spacer binds a chiral amino
acid-type selector via the carboxyl group, the other
two functions easily react with the amino group of
the selector. The epoxy-activated and L-proline incorporating silica was the Rrst chiral-bonded stationary
phase on the market (Chiral ProCu, Serva, Heidelberg, Germany). Silica-bonded ligand exchangers
have been used successfully to resolve racemates of
numerous amino acids, hydroxy acids, N-dansyland N-alkyl-amino acids, -triSuoromethyl--amino
acids, dipeptides, some -amino acids, etc. Amino
alcohols do not form stable chelates with Cu(II) ions,
but are easily resolved after conversion into Schiff
bases or, better still, after reaction with bromoacetic
acid, which converts the amino group into a chelating
glycine moiety.
The silica-bonded phases, however, still require
elevated temperatures (usually 503C) to generate sufRcient column plate numbers. In addition only neutral or weakly acidic aqueous}organic eluents, in
particular, in the pH range between 6.0 and 4.0 can
be applied. Therefore, a much more hydrolytically
stable packing was introduced by multiple-point
binding of chloromethylated polystyrene chains to
the silica matrix, followed by substituting the active
chlorine atoms of the polymer for chiral selectors.
This type of polymer-bonded phase can safely be used
at elevated temperatures, and display high resolving
power towards numerous racemates, as exempliRed
in Figure 1. In LEC, the elevated temperature does
not noticeably affect the enantioselectivity of the
column.
III / CHIRAL SEPARATIONS / Ligand Exchange Chromatography
2373
Chiralpak WH and WM (Daicel, Japan), TSK gel
Enantio L1 (Toso, Japan), MCI gel CRS 10W
(Japan), Chirosolve L-proline, Chirosolve L-valine
(JPS Chimie, Switzerland). Any scientiRc evaluation
of results obtained by using these phases is, however,
complicated, since the exact structure of immobilized
chiral ligands on many commercially available chiral
stationary phases is not speciRed by the manufacturers.
Chiral Dynamically Coated Stationary
Phases
Figure 1 Enantiomeric resolution of a mixture of eight DL-amino
acids with a chiral stationary phase, polystyrene grafted on silica
gel and substituted with L-Hypro. Conditions: column, 250;4 mm
i.d, dp 5 m: eluent, 0.5 mM copper acetate, 10 mM ammonium
acetate, pH 4.5, acetonitrile 30%; flow rate, 0.7 mL min\1; temperature, 753C; UV 254 nm. (Reprinted with permission from
Davankov VA (1989) Chapter 15, Figure 5, p. 464. In: Krstuloviz
AM (ed.) Chiral Separations by HPLC. Applications to Pharmaceutical Compounds, pp. 446I475. Chichester, England: Ellis
Horwood.)
When dealing with complicated analyte matrixes,
it is often useful to combine in sequence a chiral
column with a conventional achiral one, in order to
supplement the resolution of enantiomeric pairs in
the chiral column with the selectivity towards different analytes of the second column. Thus, chiral ligand
exchanging bonded phases are easily compatible with
achiral reversed phase columns, since both columns
can be operated in a gradient mode with aqueous}organic eluents which contain about 0.1 mM
copper acetate and about 0.25 M ammonium acetate.
Several silica-bonded and polymeric chiral ligand
exchangers are commercially available, e.g. Chiral-Si
100 L-ProCu, Chiral-Si 100 L-ValCu, Chiral-Si 100
L-HyProCu (Serva, Heidelberg, Germany), Nucleosil
Chiral-1 (Macherey-Nagel, Dueren, Germany),
Ligand exchanging columns of an exceptional efRciency and enantioselectivity can be easily prepared
from commercially available reversed-phase columns
by dynamically coating the column with a hydrophobic chiral selector. According to the technique suggested by Davankov and Unger in 1980, percolating
a solution of N-alkyl-L-hydroxyproline in aqueous
methanol through the column results in depositing
the chiral selector on the bonded alkyl stationary
phase. After a subsequent saturation of the retained
ligand with copper ions, the column is ready for
racemate resolutions in water-rich eluents which do
not desorb the chiral selector. Depending on the
length of the N-alkyl group (heptyl, decyl or hexadecyl) and conditions of coating, the amount of the
chiral selector deposited can be easily regulated over
a very broad range. Such columns (also commercially
available from Regis Technologies, USA, as ‘Davankov columns’) display a high resolving power toward
numerous amino acids (Figure 2) and their derivatives, racemic glycyl-di- and tripeptides, 3-amino-caprolactam, as well as norephedrine and its
analogous amino alcohols. Interestingly, the N-alkylL-hydroxyproline-coated columns,
under severe
overloading conditions, show a peculiar distortion of
elution proRles of the enantiomers in that the Rrst
peak has no tail whereas the second peak shows
a very sharp front, which enormously increased the
column loadability, implying high preparative potential of the system.
Coating reversed-phase columns with N,N-dioctylL-alanine has proved especially efRcient in resolving
racemic -hydroxy acids. Chiral coatings prepared
with N?-decyl-L-histidine, Nim-decyl-L-histidine, N2octyl-(L)-phenylalaninamide, N,S-dioctyl-N-methyl(D)-pencillamine, hydrophobic Schiff bases of chiral
amino alcohols and N-dodecyl-N-carboxymethyl(1S,2R)-norephedrine show high efRciency and selectivity. Reversed-phases coated with N,S-dioctyl-(D)penicillamine and (R,R)-tartaric acid mono-(R)-1-(naphthyl)ethylamide are commercially available under the name Sumichiral OA-5000 and 6000 (Sumica
Chemical Analysis Service, Japan).
2374
III / CHIRAL SEPARATIONS / Ligand Exchange Chromatography
Figure 2 Enantiomeric resolution of a mixture of seven DL-amino acids with a chiral coating of N-decyl-L-Hypro on reverse phase
material. Conditions: column, 100;4.2 mm, LiChrosorb RP 18, dp 5 m; eluent, methanol}water (15 : 85), 0.1 mM Cu(II) acetate, pH
5.0; flow rate 2 mL min\1; temperature 203C, UV 254 nm; elution sequence; 1, L-Ala; 2, D-Ala; 3, L-Val; 4, L-Arg; 5, D-Arg; 6, L-Leu; 7,
L-Nleu; 8, D-Val; 9, L-Phe; 10, D-Leu; 11, L-Trp; 12, D-Nleu; 13, D-Phe; 14, D-Trp. (From Davankov VA, Bochkov AS, Kurganov AA,
Roumeliotis P and Unger KK (1980) Separation of unmodified -amino acid enantiomers by reverse phase HPLC. Chromatographia 13:
677}685, Figure 2, p. 680.)
Whereas long N-alkyl substituents of the above
chiral selectors are best suitable to bring about the
intercalation of the latter between the surface alkyl
groups of the reversed phase support, porous graphite
offers a Sat and rigid surface for the chiral selector to
stick to. In this case, polyaromatic substituents show
better retention and resolving power of the coated
chiral selector, as is the case with N-naphthylmethylL-proline or N-(2-naphthalenesulfonyl)phenylalanine
(see Figure 3).
Similarly, hydroxylated macroporous silica gel can
be coated with hydrophilic chiral selectors such as
silver(I)-d-camphor-10-sulfonate or (#)-cobalt(III)tris-ethylenediamine trichloride. In non-aqueous
media, these phases easily resolve racemates of some
chiral diene and cyclopentadienylrhodium(I)-norbornadiene complexes. Since in the latter case
ethylenediamine ligands of the inert cobalt complex
are not exchanged for any new ligands, the separation
should be ascribed to ligand exchange in the outer,
solvation shell of this complex.
Finally, polymeric chains of polyacrylamide grafted with L-proline can also be permanently adsorbed
onto the surface of silica gel, to give a chiral ligand
exchanger which resolves amino acids in polar media.
The ease of preparation of chiral-coated ligand
exchanging phases, their exceptional efRciency, durability, wide application range and the opportunity of
regulating the elution order of the enantiomers of
interest by simply changing the conRguration of the
selector applied, make the technique increasingly
popular.
Chiral Ligand Exchanging Mobile
Phases
In the technique described in the previous section, the
chiral selector permanently resides on the surface of
III / CHIRAL SEPARATIONS / Ligand Exchange Chromatography
2375
Figure 3 Enantiomeric separation of racemic amino acids and hydroxy acids with a chiral coating of N-(2-naphthalenesulfonyl)-Lphenylalanine on porous graphite. Conditions: column PGC 94F (5 m, 50;4.6 mm), eluent 2.0 mM Cu(CH3COO)2, pH 5.6, flow rate
0.5 mL min\1, UV 254 nm, temperature 303C. (Reprinted with permission from Knox JH and Wan Q-H (1995) Chromatographia 40:
9}14.)
the column packing. The coating is deposited prior to
the chromatographic experiments and no eluents
should be used which cause a leakage of the selector.
A different approach, which was independently suggested in 1979 by the groups of Karger and Lindner
and Hare and Gil-Av, consists in using chiral selectors
which predominantly reside in the mobile phase. In
this case, the selector complex has to be continuously
introduced into the column. Analytes to be resolved
in such a chiral eluant form mixed-ligand complexes
with the selector, which are diastereomeric in their
structure, and, therefore, may be differently retained
through the interaction with the column packing.
A great advantage of this technique is that many
different chiral selectors can be easily tested with
respect to the analyte to be resolved. Another advantage is that smaller ligands are usually applied as the
eluent dopants, which results in enhanced rates of
ligand exchange and better efRciency of the columns.
A disadvantage is the loss of the chiral selector and
problems of separating the selector from the enantiomers resolved in the case of preparative experiments.
It should be emphasized also that the two enantiomers separated in the achiral column enter the de-
tector cell with the chiral eluent in the form of ternary
complexes. These complexes are diastereomeric and
can signiRcantly differ in molar extinction. Indeed,
a case of enantioselective Suorescence quenching of
dansylamino acids by a chiral Cu(II) complex of Lphenylalanylamide in aqueous solutions has been reported, thus requiring two calibration curves for
a quantitative determination of the two enantiomers.
In combination with Cu(II), Zn(II), Ni(II) and some
other transition metal ions, many chiral selectors
have been shown to function successfully in reversed
phase systems as chiral dopants. They mainly belong
to the following classes of chelating compounds:
E unsubstituted
L--amino
acids
(proline,
phenylalanine, isoleucine, histidine, etc.);
E N-alky-L-amino acids (N-benzyl-proline, N,N-dipropyl-alanine, N,N-dimethyl-valine);
E L--amino acid amides (valine amide, phenylalanine amide, prolyloctylamide, aspartylalkylamide);
E esters of L--amino acids (histidine methyl ester);
E peptides (prolyl-valine, aspartylphenylalanine
methyl ester or aspartame);
2376
III / CHIRAL SEPARATIONS / Ligand Exchange Chromatography
E chiral diamine (N,N,N,N-tetramethyl-(R)-propanediamine-1,2-( L )-alkyl-4-octyl-diethylenetriamine);
E mandelic acid;
E derivatives of (R,R)-tartaric acid (tartaric acid
mono-n-octylamide);
E nucleotides (adenosine diphosphate, -nicotinamide, adenine dinucleotide and, in particular, Savine
adenine dinucleotide).
mers. This approach is useful in analysing enantiomeric impurities in chiral compounds, where it is
always advisable to have the trace component eluting
before the major enantiomer.
Finally, it is worth mentioning that the chiral mobile phase mode of LEC is very convenient to prove
the reciprocity relations in chiral recognition, where
the selector is exchanged for the analyte and vice
versa. The enantioselectivity of the system should
remain the same, unless some additional factors interfere with the essential interactions within the diastereomeric sorption complexes.
Mainly racemic -amino acids and their numerous
derivatives, -hydroxy acids and amino alcohols are
successfully resolved. Less common, but nevertheless
successful, are resolution of substituted pterins,
phenylhydantoins and ornithine analogues.
Depending on the hydrophobicity of the analyte
and chiral selector applied, the content of the organic
modiRer in the chiral mobile phase has to be adjusted
in such a manner that the retention of enantiomers
remains in the desired time window. In addition to
this parameter, retention (and enantioselectivity) in
LEC would slightly decrease with the rise in the ionic
strength of the eluent and temperature of the column.
Conversely, rising pH and concentration of the chiral
additive in the mobile phase cause a clear increase in
retention and a less marked increase in enantiomeric
resolution.
Under normal phase conditions, with bare silica gel
as the packing, copper complexes of N,N,N,Ntetramethyl-(R)-propane-1,2-diamine can be used to
resolve amino acids and mandelic acid. Bis(Lprolinato)copper is efRcient even in combination with
sulfonated polystyrene cation exchangers, which additionally demonstrates the versatility of the chiral
mobile phase approach.
The high efRciency of the method is exempliRed by
Figure 4.
Of many enantioselective chromatographic systems
developed thus far, ligand exchanging systems are the
most examined. Understanding of the mechanism of
chiral discrimination is greatly facilitated here by the
fact that, of the three interactions needed for the
chiral selector to recognize the solute enantiomers,
two are well deRned as the trans-coordination bonds
of pairs of carboxylic and amino groups in the main
coordination square of the Cu(II) ion. The third interaction should be looked for in the out-of-plane sterical interactions or coordination in remote axial positions of the coordination sphere. Thus with L-proline
or L-hydroxyproline incorporating polystyrene-type
resins, retention of L-isomers of amino acids is diminished by sterical interactions with the water molecule
coordinated in one of the axial positions. On the
other hand, the binding of D-isomers is facilitated by
favourable hydrophobic interaction between the solute -substituent and the polystyrene matrix, as
shown below:
It is easy to imagine that, by using a mobile phase
with a chiral selector of the opposite conRguration,
we reverse the elution order of the resolved enantio-
Tridentate amino acids, L-His, L-Asp, L-Glu, LaHyp, manage to replace the water molecule from the
axial position and form one additional coordination
Mechanisms of Chiral Discrimination
in LEC
III / CHIRAL SEPARATIONS / Ligand Exchange Chromatography
2377
bond, which logically explains the inverted elution
order of these solutes:
shown above for the Rrst resin. Conversely, with
an L-phenylalanine-type chiral selector in a poly-
With tridentate L-allo-hydroxyproline as the chiral
selector, both bifunctional and trifunctional solutes
can form two coordinate bonds only and elute in the
order L before D without exception:
acrylamide matrix, the balance of steric interactions
results in a reduction in the retention of L-amino acids
(see above).
The decisive role of interactions with the
achiral sorbent matrix in the enantiomer recognition was discovered by Davankov in chiral-coated
and chiral mobile phase reversed phase systems.
Here, only one of two solute enantiomers can realize favourable hydrophobic interactions with the
silica immobilized alkyl groups, thus forming
the stronger retained diastereomeric sorption
complex.
With the L-Pro type selectors immobilized on
polyglycidylmethacrylate, polyacrylamide, and polyvinylpyridine matrixes, which occupy three coordination positions of Cu(II), the steric repulsion
interactions diminish retention of the D-amino acid
isomers as compared to L-isomers, as this was
The mechanism of chiral discrimination is less clear
with selectors and/or analytes other than -amino or
-hydroxy acids, and metal ions other than Cu(II),
since the coordination mode of ligands in these complexes is less well known.
2378
III / CHIRAL SEPARATIONS / Ligand Exchange Chromatography
Other Chromatographic and Related
Techniques of Resolving Enantiomers
in Ligand Exchanging Systems
Figure 4 Enantiomeric separation of R,S-hydroxy acids with
a chiral mobile phase. Conditions: 8 mM L-PheNH2, 4 mM
Cu(CH3COO)2, pH 6.0, ambient temperature, column Spherisorb
3 ODS-2 (3 m, 150;4.6 mm), flow rate 1 mL min\1, postcolumn
derivatization with Fe(CIO4)3, UV 436 nm. (Reprinted with permission from Galaverna G, Panto F, Dossena A, Marchelli R and
Bigi F (1995) Chirality 7: 331}336. Wiley-Liss, Inc., a subsidiary of
John Wiley & Sons, Inc.)
In gas chromatography, ligand exchange has acquired
great importance since Schurig introduced metal
complexes of chiral terpeneketonate (acylated camphor, carvone, pulegone, menthone) as additives to
stationary phase in capillary GC in 1977. Ni(II),
Mn(II), Cu(II), Zn(II), and Rh(I) proved especially
useful in this ‘complexation’ chromatography. The
resolution mechanism is ascribed in terms of the interaction between the above sterically or electronically unsaturated metal chelates with volatile ligands
which offer lone electron pairs to form a labile mixedligand sorption complex. Enantioselectivity of this
process is small, but, due to the extremely high plate
numbers attainable with capillary GC columns, excellent resolution is achieved for many classes of
alkenes, alcohols, ethers, oxirane, sulfur-containing
compounds, aziridine, etc. It is important that even
compounds containing one single electron-donating
atom (O, N, S) are successfully resolved by GC, contrary to LC, where the presence of two donors to
form a chelate with the central metal ion is highly
desirable.
Similar bonded chiral selectors, e.g. polysiloxaneanchored Ni(II)-bis-[(3-heptaSuorobutanoyl)-(1R)camphorate], Chirasil-Nickel, can also be used in the
supercritical Suid chromatography (SFC) mode. Although SFC cannot compete with GC with regard to
efRciency, a signiRcant increase in selectivity, due to
a substantially lower temperature of the column, can
compensate for the loss in plate number. Because of
the high solvation strength of supercritical carbon
dioxide, a number of low-volatility racemic alcohols,
diols and esters have been resolved by ligand exchange SFC at temperatures as low as 40}703C.
In capillary electrophoresis (CE) and other electromigration techniques, chiral ligand exchange can
be involved in several ways. Cu(L-His)2 has just been
added to the ammonium acetate electrolyte to induce
chiral resolution of dansylated amino acids. Analyte
enantiomers which are strongly involved in the
formation of positively charged mixed complexes
with Cu(L-His) move toward the cathode at a higher
rate than the uncomplexed ligands. Since the enantioselectivity of the ligand exchange reaction in the
bulk solution is small, the resolution of enantiomers
is also small. It is only slightly better with copperaspartame as chiral additive. A signiRcant improvement in selectivity can be obtained by adding
tetradecyl sulfate, as a micelle-forming surfactant.
Obviously, the enantioselectivity increases through
the interaction of the diastereomeric ternary complexes
III / CHIRAL SEPARATIONS / Ligand Exchange Chromatography
2379
nique, ligand exchange opened enormous possibilities
for chiral thin-layer chromatography. MachereyNagel in cooperation with Degussa (Germany) developed reversed-phase plates coated with copper
complexes of N-(2-hydroxydodecyl)-L-hydroxyproline in the manner described earlier by Davankov for
chiral coating of RP-columns. The Chiralplate威 has
proved to be extremely versatile in the resolution of
racemic -amino acids, their N-methyl-, N-formyl-,
-alkyl-, and halogenated amino acids, dipeptides,
-hydroxy acids, thiazolidine derivatives, anomers of
several nucleobases, etc. Copper complexes of N,Ndiallky amino acids have also been tested as chiral
coatings of plates, but so far have not found broad
application.
Finally, enantiomeric separation of phenylalanine
and lactic acid by diffusion through a liquid membrane was studied, using a solution of N-decyl-Lhydroxyproline copper complexes in hexanol}decane
mixtures as the membrane. The rate of migration of
the D-Phe was found to be about 2.4 times faster as
compared to those of the L-isomer. Though the productivity potential of the technique is high, the above
value of the kinetic enantioselectivity is still insufRcient for practical use.
Applications
Figure 5 Electrophoregram of the enantiomer separation of
DL-Phe, DL-Trp and DL-MeTrp with and without SDS. Conditions:
(a) 80 mM L-Hypro, 40 mM Cu(II) sulfate, pH 4.0; (b) 50 mM
L-Hypro, 25 mM Cu(II) sulfate, 15 mM SDS, 3 M urea, pH 4.0.
Capillary: fused silica, 75 cm;75 m i.d. (effective length, 66 cm)
U"27 kV, UV 208 nm, ambient temperature. (Reproduced with
permission from Schmid MG and Guebitz G (1996) Enantiomer 1:
23}27. Gordon and Breach Publishers.)
with the surface of the pseudo-stationary micelle
phase. Good resolution for dansyl (DNS) amino acids
has been obtained with mixed-micelle solutions containing sodium dodecyl sulfate (SDS) and copper
complexes of N,N-didecyl-L-alanine. Figure 5 shows
an example of CE resolution of underivatized racemic
amino acids with Cu(L-Hyp)2 in the support electrolyte, which is facilitated by SDS micelles. Note the
inversion of the elution order of the enantiomers and
solutes on transition from the homogeneous support
electrolyte solution to the pseudo-heterogeneous
micellar system.
In addition to paper chromatography which represents the oldest chiral planar chromatography tech-
Two principal advantages of chiral LEC should be
mentioned with respect to practical application of the
method. Firstly, the detection of compounds which
do not absorb in the near UV region is greatly facilitated by the fact that they partially elute from the LEC
column in the form of metal complexes. Even having
no strong chromophore in the molecule, they strongly
absorb at 254 nm in the complexed form. When complexed to Fe(III), hydroxy acids can be selectively
detected at 420 nm. The second great advantage of
ligand exchange is that enantioselectivity of the separation is largely unaffected by the temperature of the
column and the presence of buffer salts and organic
modiRer in the eluent. This makes the method easily
compatible with numerous other separation techniques, including multidimensional and multicolumn
chromatography.
In general, the efRciency and easiness of the direct
resolution of amino acids and hydroxy acids, without
any prior derivatization of the analyte, make LEC
a technique of choice for serial determinations of
enantiomeric compositions. This is the case, for
example in fossil dating where, depending on the
rate of racemization of the amino acid under investigation, the dating can be performed in the time
range from several hundred years to one million
years. Another application area of this kind is the
2380
III / CHIRAL SEPARATIONS / Ligand Exchange Chromatography
determination of D-amino acids in food products
caused by partial racemization through heat or
microwave treatment, and to fermentation processes.
Occurrence of D-amino acids in biological Suids
(e.g. serum, cerebrospinal Suid, urine), bone and tissues is also of great importance from a diagnostic
point of view. Rapid enantiomeric analysis techniques are needed when asymmetric synthesis or
chemical or enzymic racemate resolution processes
are being developed. Examination of the enantiomeric composition of hydroxy acids, in particular
malic acid, helps to investigate adulteration of apple
juice and other soft drinks. Rapid preparation of
enantiomers of compounds labeled with highly radioactive atoms, where only dilute solutions can be handled, is another excellent application area for chiral
LEC.
Three decades of the existence and intensive development of enantioselective LEC have shown this
technique to be an extremely versatile and productive
general approach to resolving racemates of compounds that form complexes with metal ions. Other
types of molecular interactions have been later found
to provide alternative successful ways of organizing
the formation of labile diastereomeric adducts with
appropriate chiral selectors, e.g. formation of inclusion complexes of analytes with cyclodextrins and
cyclic antibiotics or formation of charge-transfer
complexes between electron-donating and electrondeRcient aromatic groups in Pirkle-type chiral stationary phases. Designing novel chiral selectors
which simultaneously offer to the analyte several different modes of interactions could probably result in
developing chiral chromatographic systems with
wider application areas or higher enantioselectivity
with respect to particular important racemates. Thus
Rrst attempts of providing cyclodextrins with a copper complexed residue moiety have recently been
reported in the literature. Another practically unexplored Reld for further development could be ligand
exchange in nonaqueous and even nonpolar media.
With the solvent molecules not competing for the
coordination positions of the metal ion, much weaker
electron donors than carboxylic or amino functional
group of the analyte, should sufRce for the complex
formation. Ether, sulRde and carbon}carbon double
bonds should thus be suitable for complexation.
Achievements of complexing gas chromatography
and ‘argentation’ thin-layer chromatography corroborate the feasibility of this development of
ligand exchanging enantioselective column liquid
chromatography.
See also: II/Chromatography: Liquid: Chiral Separations in Liquid Chromatography: Mechanisms.
II/Chromatography: Thin-Layer (Planar): Ion Pair ThinLayer (Planar) Chromatography. III/Amino Acids and Derivatives: Chiral Separations. Chiral Separations: Capillary Electrophoresis; Cellulose and Cellulose Derived
Phases; Chiral Derivatization; Cyclodextrins and Other
Inclusion Complexation Approaches; Ion-Pair Chromatography; Liquid Chromatography; Molecular Imprints as Stationary Phases; Protein Stationary Phases; Synthetic
Multiple Interaction (‘Pirkle’) Stationary Phases; ThinLayer (Planar) Chromatography.
Further Reading
Davankov VA (1980) Resolution of racemates by ligandexchange chromatography. Advances in Chromatography 18: 139}195.
Davankov V (1989) Ligand exchange phases. In: Krstuloviz
AM (ed.) Chiral Separations by HPLC. Applications to
Pharamaceutical Compounds, pp. 446}475. Chichester,
England: Ellis Horwood.
Davankov VA (1992) Ligand-exchange chromatography of
chiral compounds. In: Cagniant D (ed.) Complexation
Chromatography, pp. 197}245. New York: Marcel
Dekker.
Davankov VA (1994) Chiral selectors with chelating properties in liquid chromatography: fundamental reSections
and selective review of recent developments. Journal of
Chromatography A 666: 55}76.
Davankov VA and Kurganov AA (1983) The role of achiral
sorbent matrix in chiral recognition of amino acid
enantiomers in ligand-exchange chromatography.
Chromatographia 17: 686}690.
Davankov VA, Kurganov AA and Bochkov AS (1983) Resolution of racemates by high-performance liquid
chromatography. Advances in Chromatography 22:
71}166.
Davankov VA, Navratil JD and Walton HF (1988) Ligand
Exchange Chromatography. Boca Raton, Florida: CRC
Press.
Gil-Av E, Tishbee A and Hare PE (1980) Resolution of
underivatized amino acids by reversed phase chromatography. Journal of the American Chemical Society 102:
5115}5117.
Guenther K (1988) Thin-layer chromatographic enantiomeric resolution via ligand exchange. Journal of
Chromatography 448: 11}30.
Schurig V (1988) Enantiomer analysis by complexation gas
chromatography: scope, merits and limitations. Journal
of Chromatography 441: 135}153.